Control of methane production by methanogenic bacteria in ruminant animals has important agronomic impact. Use of inhibitors to control the methane produced by ruminants has been recognized as a part of the mechanism for feed efficiency that results when mixed with cattle feed for both dairy and meat production. An effective additive to boost ruminant feed efficiency is a well-established part of the agronomic practice for commercial ruminant farming.
Methanogenic bacteria form methane by an anaerobic process. The group comprises the genera Methanococcus, Methanobacterium, Methanosarcina, Methanobrevibacter, Methanothermus, Methanothrix, Methanospirillum, Methanomicrobium, Methanococcoides, Methanogenium and Methanoplanus.
Inhibitors of methanogenesis in rumen perform two important functions. Cows and sheep lose 5-10% of their caloric intake to the formation of methane and the resulting loss of a carbon molecule that could have been incorporated in short chain fatty acid production. Inhibition of methane will, therefore, have a direct effect on the formation of short chain fatty acids in the rumen. Other investigators have reported the positive effect of inhibiting methane in rumen fermentation (C. J. Van Nevel, D. I. Demeyer, Manipulation of rumen fermentation, In: The Rumen Microbial Ecosystem, P. N. Hobson, and (Ed) Elsevier Publishing Co. (1988)).
Methane inhibitors have previously been developed for feedstock additives to increase feed efficiency. The inhibitors fall generally into two classes. The first class induces those that affect methane formation indirectly by interfering with the electron flow upstream of the methanogen in the microbial food chain. Examples of this group would be nitrates and nitrites. The second class includes those that affect methanogens directly. Examples of such compounds are ionophores, antibiotics, and polycyclic quinones. Ionophores include, for example, RUMENSIN(copyright) (monensin sodium), lasalocid A, salinomycin, avoparcin, aridcin, actaplanin, and penicillin. A more complete list is cited in: C. J. Van Nevel, D. I. Demeyer, Manipulation of rumen fermentation, In: The Rumen Microbial Ecosystem, P. N. Hobson, and (Ed) Elsevier Publishing Co. (1988). Polycyclic quione activity in this regard are referenced in U.S. Pat. No. 5,648,258 (Odom).
The inhibition of methane in rumen by polycyclic quinones (PCQ) operates by a different mechanism than ionophores. PCQ""s are redox catalysts that block reduction of electron receptors at the cytochrome c-3 site in the cell wall of anaerobic bacteria, such as methanogens and sulfate reducers. Weimer reveals the action of 9,10-anthraquinone in U.S. Pat. No. 5,385,844 as it applies to reducing sulfate by sulfate reducing bacteria.
Ionophores act as antibiotics with the result that target bacteria concentrations in the rumen are reduced. Since 9,10-anthraquinone does not reduce target bacteria concentration in the rumen, the two mechanisms are clearly distinct.
Garcia-Lopez et al. has demonstrated the use of PCQ""s and ionophores each separately can reduce biogenic methane. (P. M. Garcia-Lopez, L. Kung, Jr., J. M. Odom xe2x80x9cIn Vitro Inhibition of Microbial Methane Production by 9,10-anthraquinonexe2x80x9d. Journal of Animal Science 1996, 74:2276-2284).
In its primary aspect, the invention is directed to a synergistic method for reducing methane formation in the rumen of ruminants comprising administering to the ruminant at least one ionophore compound, and at least one polycyclic quinone compound.
As used herein, the term xe2x80x9crumenxe2x80x9d refers to the gastrointestinal section found in ruminants (i.e. cattle, deer, moose, camels, sheep, goats, oxen, water buffalo, and musk oxen) where food is partially digested through bacterial fermentation.
A. In General
It is recognized that the administration of an ionophore compound or the administration of a polycyclic quinine (PCQ) to a ruminant will reduce methane and boost feed efficiency in the ruminant. However, applicant has discovered that when the two classes of compounds (ionophores and PCQ""s) are administered simultaneously to a ruminant, a synergistic reduction of methane occurs. The advantage of employing this technique is to provide additional feed efficiency for agronomic benefits in ruminant raising. In addition, the levels of antibiotics in feed can be reduced which helps lower the adaptive challenge by non-target bacteria in the rumen and, thereby, lessens the likelihood of adaptation and resistance by rumen bacteria to the antibiotic.
B. Polycyclic Quinones (PCQ""s)
A wide variety of polycyclic quinones can be used in the invention. As used herein, the term xe2x80x9cpolycyclic quinonexe2x80x9d or xe2x80x9cPCQxe2x80x9d refers to bicyclic, tricyclic and tetracyclic condensed ring quinones and hydroquinones, as well as precursors thereof. On the whole, the non-ionic polycyclic quinones and polycyclic hydroquinones (herein referred to collectively as PCQ""s) have very low solubility in water at ambient temperatures. For use in the invention, it is preferred that such PCQs have water solubility no higher than about 1000 ppm by weight.
In addition, as noted above, certain precursors of such PCQ""s can also be used in the invention either combined with the relatively insoluble PCQ""s or by themselves. Such precursors are anionic salts of PCQ""s, which are water soluble under alkaline anaerobic conditions. However, these materials are not stable and are easily converted to the insoluble quinone form upon exposure to oxygen.
Among the water-insoluble PCQ""s, which can be used in the invention, are anthraquinone compounds. As used herein, the term xe2x80x9canthraquinonexe2x80x9d or xe2x80x9cAQxe2x80x9d refers to 9,10-anthraquinone, naphthoquinone, anthrone (9,10-dihydro-9-oxo-anthracene), 10-methylene-anthrone, phenanthrenequinone and the alkyl, alkoxy and amino Derivatives of such quinones, 6,11-dioxo-1H-anthra[1,2-c]pyrazine, 1,2-benzanthraquinone, 2,7-dimethylanthraquinone, 2-methylanthraquinone, 3-methylanthraquinone, 2-aminoanthraquinone and 1-methoxyanthraquinone. Of the foregoing cyclic ketones, 9,10-anthraquinone and methylanthraquinone are preferred because they appear to be more effective. Naturally occurring anthraquinones can be used as well as synthetic anthraquinones.
xe2x80x9cAnthraquinonexe2x80x9d or xe2x80x9cAQxe2x80x9d compounds can further include insoluble anthraquinone compounds, such as 1,8-dihydroxy-anthraquinone, 1-amino-anthraquinone, 1-chloro-anthraquinone, 2-chloro-3-carboxyl-anthraquinone, 1-hydroxy-anthraquinone and unsubstituted anthraquinone. Various ionic derivatives of these materials can be prepared by catalytic reduction in aqueous alkali.
In addition, a wide variety of anthrahydroquinone compounds can be used in the method of the invention. As used herein, the term xe2x80x9canthrahydroquinone compoundxe2x80x9d refers to compounds comprising the basic tricyclic structure, such as 9,10-dihydroanthrahydroquinone, 1,4-dihydroanthrahydroquinone, and 1,4,4a,9a-tetrahydroanthrahydroquinone. Anthrahydroquinone itself is 9,10-dihydroxyanthracene.
More particularly, both water-insoluble and water-soluble forms can be used. The non-ionic compounds are largely insoluble in aqueous systems, while ionic derivatives, such as di-alkali metal salts, are largely soluble in water. The water-soluble forms are stable only in high pH anaerobic fluids. Low pH fluids (pH less than about 9-10) will result in the formation of the insoluble molecular anthrahydroquinone. Aerobic solutions will incur oxidation of the anthrahydroquinones to anthraquinone. Thus, anthrahydroquinones will not exist for long periods of time in an aerated environment. For these reasons, anthrahydroquinone treatments are usually implemented with the soluble ionic form in a caustic solution. Sodium hydroxide solutions are preferred over the hydroxides of other alkali metals for economic reasons. Rumen physiology may limit the pH of such a preparation, but use of sodium hydroxide in ruminant feed is an established practice.
The extraordinary effectiveness of various forms of anthraquinone lies in their non-reactivity. These products are transported into the biofilm, diffuse through the biofilm voids, and then diffuse or are randomly transported by Brownian motion into the bacterial microcolonies without reduction in concentration as a consequence of a exopolysaccharide matrix present in the biofilm.
Even though solid particles of polycyclic quinone (PCQ) are required to inhibit the methane-producing bacteria, the PCQ can be introduced into the microbial environment in several physical forms. The PCQ can be introduced as a dispersion of these solid particles throughout the feed at the appropriate dose. The ionic (sodium salt) form of the PCQ will allow it to be solubilized in an anaerobic caustic solution as long as the pH is greater than 12 and preferably greater than 13. The salt stays soluble if the pH of the solution remains above about 12, with precipitation of solid PCQ taking place as the pH is reduced below this value. In the soluble form or with a slight amount of precipitated PCQ (typically in colloidal form), anthraquinone is in molecular form or consists as extremely small (submicron-sizes) particles. When the PCQ added to the water is in the form of a suspension of finely divided particles, it is preferred that their largest dimension be no greater than 50 micrometers, and preferably no greater than 5-10 micrometers so that they can more easily pass through biofilm.
Whether the soluble or insoluble anthraquinone is used, it has been observed that the functional attachment of the anthraquinone particles to the bacteria is limited in time by metabolism of the particles by the sulfate-reducing bacteria. Thus, application of the treating medium must be repeated periodically in order to maintain inhibition effectiveness.
Unlike antibiotics, which are lethal to rumen based bacteria, especially methanogens, PCQ""s are non-lethal in their mechanism. Studies by Cooling et al. have revealed the mechanism of action of anthraquinones in sulfate-reducing bacteria (F. B. Cooling III, C. L. Maloney, En. Nagel, J. Tabinowski and J. M. Odom. xe2x80x9cInhibition of Sulfate Respiration by 1,8-Dyhydroxy-Anthraquinone and other Anthraquinone Derivativesxe2x80x9d. Applied And Environmental Microbiology, August 1996, p. 2999-3004). PCQ""s block the production of adenosine triphosphate by the bacteria and thereby inhibit respiration using sulfate as an electron acceptor. The sulfate-reducing bacteria respire by alternate mechanisms under these conditions and are not killed. SRBs and methanogens are closely linked in their ecological niche in the rumen and other anaerobic environments. The PCQ effect on methanogens is either a direct effect similar to the SRB mode of action or indirect since methanogens are dependent on SRB for micro-nutrients. In both conditions, methanogens thrive in the presence of PCQs without forming the normal levels of methane.
C. Ionophores
Compounds known as ionophores are generally defined as substanced that facilitate transmission of an ion, (such as sodium), across a lipid barrier such as a cell membrane. Two ionophore compounds particularly suited to this invention are the RUMENSIN(copyright) (monensin sodium) product from Eli Lilly which is a sodium salt of a complex molecule of the general formula C36H61011NA (formula weight 692.9) and lasalocid A from Hoffman LaRoche. Other ionophore compounds are discussed in the Background Section of this application, and include salinomycin, avoparcin, aridcin, actaplanin and penicillin among others. In the rumen, ionophores act as effective antibacterial agents. Killing methane producing bacteria in the rumen of cattle decreases the loss of carbon from the rumen fluid as methane which is a similar action to AQ.
Inhibition of methane by ionophores follows a mode of action where methanogens and other bacteria that produce pure hydrogen and carbon dioxide are reduced in concentration. The antibacterial action of ionophores is the direct cause of the reduction in methanogenesis (P. M. Garcia-Lopez et al., 1996 In Vitro Inhibition of Microbial Methane Production by 9,10 anthraquinone: Delaware Agricultural Experimental Station, paper no. 1567). Reduction in bacteria concentration in the rumen can also affect other microlife that is generally helpful in rumen digestion and the formation of short chain fatty acids. The short chain fatty acids are the source of energy required by ruminants. Increases in concentrations of proprionate and sometimes butyrate are accompanied by reductions in acetate in rumen affected by ionophores. Ionophores tend to lower concentrations of bacteria that produce hydrogen, which is contrary to the results seen with PCQ""s. Hydrogen values tend to increase with PCQ""s, which should lead to stimulation of bacteria levels that process hydrogen into butyrate. (B.fibrisolvens). Acetate forming bacteria are also reduced with ionophores where PCQ""s would tend to stimulate the formation of more acetate if acetogenic bacteria such as (Acetitomaculum ruminis)2 (Greening and Leedle, 1989 Enrichment and Isolation of Acetitonaculum Rumninis gen.nov.sp. Nov; Acetogenic Bacteria from the Bovine Rumen. Arch. Microbial. 151:399) are present. The advantage of increased bacterial formation of short chain fatty acids is a boost in the food value of the feed ruminants.
D. Methods of Operation
The function of the PCQ is to act as an inhibitor specific for methanogens and sulfate reducers found naturally in rumen fluid. Anthraquinone (AQ) is the preferred PCQ to be used in the invention. The inhibition of methane by AQ is a separate and distinct mechanism from the antibiotic effect of an ionopbore compound, such as monensin sodium. Bacteria counts of methanogens are not affected by 9,10-anthraquinone while ionophores reduce the viability of methanogens. Therefore, the actions of the two classes of compounds are distinct and an additive effect would be expected. Contrary to expectations, the results show synergistic effects.
The customary method of adding a feed additive is to premix the compound with a binder and a carrier so that the premix carries a diluted concentration of active ingredient. The premix is blended with the rations for the animal in a subsequent process so that there is a certified final concentration of active ingredient in the feed. A further method of adding PCQ to animal rations would be a direct admixture of active ingredient with the rations by means of a liquid formulation sprayed onto the feed or by a dry formulation admixed by blending. The use of a sodium salt of anthraquinone in a high pH medium could also be used as a way to enhance the distribution of AQ in animal feed. Certain feeds would have nutritive improvement due to the delignification of the fibers caused by the well known action of a high pH medium and the catalytic action of AQ on the lignin bonds that make fiber less digestible.
The preferred concentration of ionophores such as monensin sodium, 2,2-dichloracetamide is preferably in the range of 0.5 ppm-35 ppm and more preferably in the range of 5-10 ppm in the rumen fluid of the ruminant. AQ is preferably in the range of 10-500 ppm and more preferably in the range of 10-100 ppm in the rumen fluid of the ruminant.